The immune response comprises a cellular response and a humoral response. The cellular response is mediated largely by T-lymphocytes (alternatively and equivalently referred to herein as T-cells), while the humoral response is mediated by B-lymphocytes (alternatively and equivalently referred to herein as B-cells).
B-cells produce and secrete antibodies in response to the presentation of antigen and MHC class II molecules on the surface of antigen presenting cells. Antigen presentation initiates B-cell activation with the engagement of the B-cell receptor (BCR) at the cell's surface. Following engagement, the BCR relays signals that are propagated through the cell's interior via signal transduction pathways. These signals lead to changes in B-cell gene expression and physiology, which underlie B-cell activation.
T-cells produce costimulatory molecules, including cytokines, that augment antibody production by B-cells during the humoral immune response. Cytokines also play a role in modulating the activity of T-cells themselves. Many T-cells act directly to engulf and destroy cells or agents that they recognize by virtue of the cell surface proteins they possess. The engagement of cell surface receptors on T-cells results in the propagation of intracellular signals that provoke changes in T-cell gene expression and physiology, which underlie the cellular immune response.
Antigen recognition alone is usually not sufficient to initiate a complete effector T- or B-cell response. The generation of many B-cell responses to antigen is dependent upon the interaction of B-cells with CD4+ helper T-cells directed against the same antigen. These helper T-cells express CD40L (CD154) which binds to the cell surface receptor, CD40, on resting B-cells. This interaction provides a critical activation signal to B-cells. Mutations in the CD40L lead to the X-linked immunodeficiency disorder hyper-IgM syndrome, which is characterized by low levels of IgA and IgG, normal to elevated levels of IgM, absence of germinal center formation, and decreased immune response. In addition, transgenic mice lacking CD40 exhibit reduced graft rejection. (Zanelli et al., Nature Medicine, 6: 629–630, 2000; Schonbeck et al., Cell Mol Life Sci, 58:4–43, 2001).
Non-lymphocyte myeloid derivatives are also activated by surface receptor engagement in immune response and in response to injury. For example, mast cells and basophils are activated by binding of antigen to surface IgE, while platelets are activated by the binding of thrombin to its receptor.
Intercellular communication between different types of lymphocytes, as well as between lymphocytes and non-lymphocytes in the normally functioning immune system is well known. Much of this communication is mediated by cytokines and their cognate receptors. Cytokine-induced signals begin at the cell surface with a cytokine receptor and are transmitted intracellularly via signal transduction pathways. Many types of cells produce cytokines, and cytokines can induce a variety of responses in a variety of cell types, including leukocytes. The response to a cytokine can be context-dependent as well as cell type specific.
Dysregulation of intercellular communication can perturb leukocyte activity and the regulation of immune responses. Such dysregulation is believed to underlie certain autoimmune disease states, hyper-immune states, and immune-compromised states. Such dysfunction may be cell autonomous or non-cell autonomous with respect to lymphocytes.
The activation of specific signaling pathways in leukocytes determines the quality, magnitude, and duration of immune responses. In response to transplantation, in acute and chronic inflammatory diseases, and in autoimmune responses, it is these pathways that are responsible for the induction, maintenance and exacerbation of undesirable leukocyte responses. Identification of these signaling pathways is desirable in order to provide diagnostic and prognostic tools, as well as therapeutic targets for modulating leukocyte function in a variety of disorders or abnormal physiological states. In addition, the ability to modulate these pathways and suppress normal immune responses is often desirable, for example in the treatment of hosts receiving a transplant.
While the extracellular domains and cognate ligands of lymphocyte receptors vary widely, many receptors have similar intracellular domains (such as the “immunoreceptor tyrosine-based activation motif” (ITAM)), and associate with common intracellular signaling molecules.
Tyrosine kinase activation is a critical event in the propagation of intracellular signals by many receptors on lymphocytes, including antigen receptors on B- and T-cells (for a review see Turner et al, Immunology Today, 21:148–154, 2000, incorporated herein in its entirety by reference).
With regard to the B-cell antigen receptor (BCR), the BCR is rapidly phosphorylated on tyrosine residues following engagement of the receptor by antigen or other crosslinking agents. This tyrosine phosphorylation leads to associations with several SH2-containing signaling proteins. SH2-containing proteins are known to bind to phosphorylated tyrosine residues in the context of specific amino acid sequences.
Many non-receptor tyrosine kinases have been shown to interact with tyrosine phosphorylated receptors in lymphocytes, including the antigen receptors of B- and T-cells. These non-receptor tyrosine kinases include members of the src family and the Bruton's tyrosine kinase (BTK) family. Importantly, many of these genes are associated with oncogenesis.
A non-receptor tyrosine kinase with an as yet undetermined role in lymphocytes is hematopoietic cell kinase (Hck). Hck is a tyrosine kinase comprising a consensus myristylation sequence, as well as SH2 and SH3 domains at the N-terminus, and a C-terminus tyrosine kinase domain possessing signature nucleotide-binding and catalytic tyrosine kinase domain motifs (Ziegler et al., Mol. Cell. Biol., 7:2276–2285, 1987; Quintrell et al., Mol. Cell. Biol., 7:2267–2275, 1987). Hck is highly expressed in cells of the myeloid lineage, particularly granulocytes. A role for Hck in the differentiation of these post-mitotic and short-lived cells has been suggested. In addition, chromosomal mapping of the Hck gene has implicated it in myeloid leukemias (Quintrell et al., supra).
HCK is also expressed in B cells, and is tyrosine phosphorylated in response to BCR stimulation. Further, in vitro kinase assays have demonstrated that HCK activity is increased in response to BCR stimulation. In addition HCK interacts with Bcr-abl, p120-Cbl, Btk, Ig-β and Ig-α, hBRAG, WASP and WIP, HIV Nef (in vitro) and the GP-130 chain of the IL-6 receptor (Zverkoczy et al., J. Biol. Chem., 275:20967–20979, 2000, and references therein; Scott et al., J. Biol. Chem., 277:28238–28246,2002 Brown et al., J. Virol., 73:9899–9907, 1999). In addition, WIP activates HCK, and WASP is an HCK substrate which is tyrosine phosphorylated by HCK.
In monocytes, Hck is upregulated and tyrosine phosphorylated in response to IL-2. Further, tyrosine kinase inhibitors inhibit Hck activation and the response of monocytes to IL-2 (Bosco et al., J. Immunol. 164:4575–4585, 2000). Hck is also responsive to CD38 and MHC II stimulation in monocytes (Zilber et al., Proc. Natl. Acad. Sci., 97:2840–5845, 2000 ).
In addition to kinases, phosphatases also play an important role in lymphocyte activation. Calcineurin is a calcium-sensitive protein serine/threonine phosphatase comprised of a catalytic and a regulatory subunit. Calcineurin is activated by the binding of Ca2+/calmodulin, and is inhibited by the immunosuppressants FK506 and cyclosporin A. Binding of cyclosporin to calcineurin blocks substrate access to the active site of the phosphatase (Liu et al., Cell, 66: 807, 1991).
The activation of calcineurin is an important regulatory step in lymphocyte activation. Calcineurin regulates phosphorylation of the nuclear factor in activated T-cells (NFAT) transcription factor, and thereby regulates nuclear import of NFAT and its ability to regulate transcription. The expression of many factors involved in lymphocyte activation, including cytokines (such as IL-2) and cell surface molecules (eg. CD40L) lies downstream of NFAT activation. (see Klee et al., J Biol Chem., 273:13367, 1998; Stankunas et al., Cold Spring Harbor Symposia Quant. Biol., 64: 505–516, 1999).